Citrus diseases caused by viruses lead to significant economical loss worldwide (Derrick, K. S. and Timmer, L. W., Annu. Rev. Phytopathol., 38:181-205 (2000)). One example of a disease causing virus is Citrus Tristeza Virus (CTV), a member of the Closterovirus group, which induces serious disease syndromes in citrus, including quick decline resulting in death of trees on sour orange rootstock, and stem pitting of scion cultivars regardless of the rootstock used (Bar-Joseph et al., Annu. Rev. Phytopathol. 27:291-316 (1989)).
In 1937, millions of sweet orange trees grafted on to sour orange rootstocks were lost in Brazil, due to citrus tristeza. The exchange of sour orange stock by Rangpur lime rootstock solved this problem (Gimenes-Fernandes, N. and Bassanezi, R. B. Summa, Phytopathologica., 27:93 (2001)).
In addition to CTV, other disease causing viruses are economically important. For example, CSDV causes citrus tree (sweet orange) death a few months after symptom detection (Gimenes-Femandes, N. and Bassanezi, R. B. Summa Phytopathologica., 27:93 (2001)). The disease is associated with the presence of a Tymovirus in symptomatic trees and it is believed to be carried by an insect vector (Maccheroni Jr. et al, Journal of Virology, 79(5):3028-37 (2005)).
Citrus leprosis (CiL) is another economically relevant virus related disease, especially in the State of São Paulo, the largest citrus producing area in Brazil, where the disease is endemic. CiL is also starting to move to Central American countries, including Guatemala, Honduras, Costa Rica and Panama (Dominguez, F. S.; Bandel, A.; Childers, C; Kitajima, E. W. Plant Disease, 85:228 (2001)). CiL is vectored by mites from the Brevipalpus genus which transmit a virus herein designated Citrus leprosis virus (CiLV), and usually affects sweet orange (Rodriguez, J. C.; Kitajima, E. W.; Childers, C. C.; Chagas, C. M. Exp and Appl Acarol, 30:161-79 (2003)). The symptoms related to this disease include circular clorotic lesions on both sides of the leaf. Eventually, the lesions become necrotic, assuming a central brownish color, leading to defoliation. Lesions are also present on branches and fruits, causing severe fruit damage and drop, leading to serious tree decline. Apart from sweet orange trees, which are very susceptible to CiL, the CiL symptoms can affect other varieties such as tangerine, but the susceptibility to the virus can vary from resistant to tolerant depending on the citrus variety. Mechanical transmission of CiLV can be achieved successfully from citrus to citrus, and from citrus to herbaceous plants (e.g., Chenopodium amaranticolor, C. quinoa and Gomphrena globosa) using lesion extracts (Colariccio, A.; Lovisolo, O.; Chagas, C. M.; Galetti, S. R.; Rossetti, V.; Kitajima, E. W. Fitopatol. Bras., 20:208-213 (1995)).
CiLV has been found in two different forms. Under electron microscopy, CiLV can be seen as rod shaped, enveloped particules, 30-40 nm×110-130 nm in size, present in the cytoplasm of leaf tissues (Kitajima, E. W.; Muller, G. W.; Costa, A. S.; Yuri, W. Virology, 50:254-258 (1972)). In another report, similarly sized and shaped particles were found in the nucleus; however these were not surrounded by an envelope (Colariccio, A.; Lovisolo, O.; Chagas, C. M.; Galetti, S. R.; Rossetti, V.; Kitajima, E. W. Fitopatol. Bras., 20:208-213 (1995)). Based on the morphology and occurrence of the two forms, CiLV has been classified as a Rhabdovirus.
Recently, Locali et al. (Plant Disease, 87:1317-1321 (2003)) were able to isolate some CiLV sequences using RT-PCR from RNA samples prepared from symptomatic leaves.
The control of CiL is currently carried out with acaricides, which exterminate the vector, Brevipalpus phaenicis; however, expenditures directed to exterminating this mite cost over 100 million dollars every year. (Rodriguez, J. C.; Kitajima, E. W.; Childers, C. C.; Chagas, C. M. Exp and Appl Acarol, 30:161-79 (2003)). This figure represents 80% of the total expenses relating to defenses against the pest. Therefore, there is the need to improve the means to control this disease that is both cost effective and efficient. Also, there is a worldwide concern to reduce the amount of toxic products, such as acaricides, which pollute the environment.
In order to explore the possibility of using alternative ways to control CiL that are economically sound and do not involve environmentally toxic compounds, a program to sequence the genome of CiLV was established. Several approaches were used to sequence the genome of this virus. Initially, short specific sequences from the Replicase (402 bp) and Movement Protein (MP) (339 bp) genes were obtained from symptomatic leaves of sweet orange, using diagnostic RT-PCR and gene specific primers MP-F (5′ GCGTATTGGCGTTGGATTTCTGAC 3′) (SEQ ID NO: 1) and MP-R (5′ TGTATACCAAGCCGCCTGTGAACT 3′) (SEQ ID NO: 2) for the Movement Protein and REP-F (5′ GATACGGGACGCATAACA 3′) (SEQ ID NO: 3) and REP-R (5′ TTCTGGCTCAACATCTGG 3′) (SEQ ID NO: 4) for the Replicase (Locali, E. C.; Freitas-Astua, J.; Takita, M. A.; Astua-Monge, G.; Antoniolli, R.; Kitajima, E. W.; Machado, M. A. Plant Disease, 87:1317-1321 (2003)). These sequences were used as templates to design primers to extend these regions and obtain more virus sequences toward the 5′ and 3′ regions of the two genes. Another approach was the construction of a subtractive library. In this technique, polyA RNA from symptomatic and asymptomatic leaves were extracted and converted into cDNA, separately. These cDNAs were hybridized to each other at specific temperature and salt concentrations and amplified by PCR in such a way that only the sequences specific to the symptomatic leaves were amplified. The remaining cDNA sequences that are common to both cDNA populations were not amplified and therefore lost. The subtracted PCR amplified sequences were cloned and sequenced to search for virus sequences. Also, the subtractive library was enriched for viral sequences using primers based on the viral sequences obtained by the RT-PCR in the first experiment described above. The combination of these 2 approaches generated the complete sequence of the CiLV genome, which is a bipartite virus, i.e., one that presents two RNA segments. The nucleotide sequence of RNA1 is 8730 bp long (SEQ ID NO: 5). One large ORF of 7539 bp, starting with an AUG codon at position 108-110 and terminating with an UAA codon at 7644-7646, was detected. This ORF encodes a 2512 amino acid replicase protein (with an estimated molecular weight of 286.4 KDa) (SEQ ID NO: 6) with sequence similarities to other replicases from plant viruses, especially the Furovirus. The following domains are present in the polyprotein: i) a methyl transferase, from residues 128 to 325; ii) a putative protease comprising amino acids 683 to 803; iii) a helicase, from amino acid 1521 to amino acid 1697 and iv) a RNA-dependent RNA polymerase domain comprising amino acid 2221 to 2458. All numbering refers to SEQ. ID. NO: 6. There is a second ORF (792 bp), in RNA1, starting at position 7709-7711 (AUG) and terminating at 8498-8500 (UAG). It encodes a putative 263 amino acid polypeptide (with an estimated molecular weight of 29.1 KDa) (SEQ ID NO: 7), herein called p29. This polypeptide has 32% identity to Sindbis virus capsid protein. RNA1 has 107 nucleotide long 5′ UTR (1 to 107) and a 230 nucleotide 3′ UTR (position 8501 to 8730), excluding its polyA tail.
The sequence of RNA2 is 4975 bps long (SEQ ID NO: 8), and contains 4 putative ORFs. The first one, closest to the 5′ end, “p15”, is 393 nucleotide long (AUG at position 67 and UAA at 459), encoding a 130 amino acid polypeptide (SEQ ID NO: 9). This polypeptide has 21% identity to an envelope glycoprotein from Human Immunodeficiency Virus-1 (Yamaguchi, Y., Delehouzee, S., Handa, H. Microbes Infect. 4, 1169-75 (2002)). The second ORF, “p61”, comprises 1614 nucleotides, starting at 1590 (AUG) and terminating at 3203 (UAA), encoding a 537 amino acid polypeptide (SEQ ID NO: 10). This polypeptide has 23% identity to a Saccharomyces cerevisiae mannosyltransferase KTR4. The third ORF (p32) is 894 nucleotide long, with its start codon at position 3228 (AUG) and stop codon at 4121 (UAA). It encodes a 297 amino acid polypeptide with an estimated molecular weight of 32.5 KDa (SEQ ID NO: 11). Similarity searches at the NCBI GenBank showed significant homology to movement proteins of plant viruses (E value=7e-09), especially those from Furovirus and Bromovirus. The last ORF in RNA2 (p24) is 645 nucleotide long. It is transcribed in a different frame than p61 and p32, and overlaps with the terminal part of the movement protein by 29 nucleotides. Its start codon is located at position 4093 and its stop codon is at position 4737 (UGA). It encodes a protein of 214 amino acids length (SEQ ID NO: 12). This polypeptide has similarity to a glycoprotein precursor (CD47-like protein) from Sheeppox Virus (Tulman, E. R., Afonso, C. L., Lu, Z, Zsak L., Sur, J. H., Sandybaev, N. T., Kerembekova, U. Z., Zaitsev, V. L., Kutish, G. F., Rock, D. L. J. Virol. 76, 6054-61 (2002)). All nucleotide references for RNA2 refer to SEQ. ID. NO: 8.
The sequence of CiLV permitted design of primers to amplify all parts of its genome in overlapping fragments, by RT-PCR (FIG. 1A). Analysis of symptomatic and asymptomatic leaves by RT-PCR (FIG. 1B) showed that only symptomatic leaves contained viral sequences. Northern blots prepared with total RNA extracted from symptomatic and asymptomatic leaves and hybridized to radioactive probes made with DNA fragments from RNA1 and RNA2 showed that only the symptomatic leaves have both 8730 and 4975 bp bands associated to CiL (FIG. 2). Also, RT-PCR made with RNA extracted from viruliferous mites and gene specific primers amplified a 1.7 kb band (FIG. 3). These results associate CiLV genomic sequence to CiL symptoms and the presence of the virus in the vector B. phoenicis. Based on these data the new virus is considered to be associated with CiL disease.